Objectives for the next year of support are: Project 1. We intend to complete immunocytochemical studies of cholinergic neurons and synapses in rat neostriatum, and finish quantitative analyses of the temporal decrease of GABAergic terminals that is manifest in a monkey model of focal epilepsy. Moreover, we will initiate studies where both cholinergic and GABAergic markers are simultaneously localized by immunocytochemistry within the cerebral cortex, neostriatum, thalamus, and basal forebrain. Project 2. Our plans include studies to: a) purify rat brain ChAT in sufficient quantities for amino acid sequence determination; b) generate new monoclonal antibodies to the enzyme surface and/or amino acid sequence. We intend to use the antibodies and amino acid sequence information for molecular cloning of the rat ChAT gene, using either the synthetic oligonucleotide approach or immunological screening of rat cDNA expression vector libraries. Project 3. An inhibitory mechanism of neurotransmission will be studied for synapses of the thick accessory neuron that terminate on crayfish stretch receptor neurons. Morphological evidence suggests that these synapses may exert a powerful influence on stretch receptor neurons, but this has never been analyzed physiologically. Inhibitory function will be studied both by stimulating the thick accessory neuron and by iontophoretic application of GABA. In addition, the relationships between the formation of synaptic vesicles and availability of transmitter will be investigated in the temperature-sensitive ChAT mutant of Drosophila, where the amount of acetylcholine in terminals can be regulated experimentally. Project 4. A search will be made among the several substances that are potent competitive inhibitors of GABA transport in the P3 fraction to obtain a substance which could serve as a covalently-linked ligand for the transporter. Upon labeling such a substance, we should be able to develop a method for the isolation of the transporter in a purified form, and to begin to study its molecular properties. We also plan to obtain monoclonal antibodies to the P3 fraction, select antibodies that inhibit the GABA transporter specifically, and employ these antibodies in affinity columns for purification of the transporter.